EARLY-STAGE BREAST CARCINOMA MECHANICALLY REMODELS THE TUMOR MICROENVIRONMENT TO PROMOTE CANCER PROGRESSION
Embargo until
2019-05-01
Date
2018-05-09
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Johns Hopkins University
Abstract
The remodeling of the tumor microenvironment is a characteristic behavior that malignant tumors utilize to progress towards late stages of cancer. Though this has been observed in in vivo studies, the measurement of tumor microenvironment remodeling in real-time and the characterization of remodeling events have not been fully studied. Here, we present an in vitro platform that can be used in concert with high-resolution confocal microscopy to capture real-time remodeling events in breast carcinoma cell lines of increasing malignancy. We carefully selected three mouse epithelial breast cell lines: EpH4-EV (normal, control), 67NR (non-metastatic, early-stage), and 4T1 (metastatic, stage IV). By embedding 3D cell cultures (spheroids) into a fabricated tumor microenvironment consisting of normal fibroblasts and rat tail type-1 collagen, extracellular matrix (ECM) remodeling was quantified in terms of collagen realignment and pore generation. Other techniques, such as traction force microscopy and fluorescence recovery after photobleaching, were employed to quantify traction stress and diffusion anisotropy due to collagen realignment. Our work demonstrates the significant remodeling events of the early-stage cancer cell line, 67NR, when compared to the control, EpH4-EV. This is quantified in terms of collagen alignment and the generation of collagen pores over time. Along with this remodeling, 67NR generates a diffusion anisotropy as molecules have directed diffusion along radially aligned collagen channels. Moreover, traction stress generation by 67NR spheroids is significantly greater than EpH4-EV, thus connecting force generation to the grade of collagen remodeling. Furthermore, culturing normal fibroblasts in the surrounding ECM results in significantly greater ECM remodeling by the embedded 67NR spheroids, suggesting that mechanical and chemical cues cause normal fibroblasts to transform into cancer-associated fibroblasts. Additionally, fibroblasts are observed to migrate towards 67NR spheroids. This result is the opposite of what occurs in previous studies, where cancer-associated fibroblasts direct metastasis by attracting cancer cells away from the primary tumor. In conclusion, early-stage, non-metastatic cancer (67NR) utilizes mechanical forces to remodel its surrounding tumor microenvironment to aid in cancer progression.
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Keywords
breast cancer, cancer, tumor microenvironment, extracellular matrix, mechanical remodeling, fibroblasts, cancer-associated fibroblasts, collagen, diffusion, traction force, confocal microscopy